Effect of Δ9-tetrahydrocannabinol on phosphorylated CREB in rat cerebellum: An immunohistochemical study

Brain Research 1048 (2005) 41 – 47 www.elsevier.com/locate/brainres

Research report

Effect of D9-tetrahydrocannabinol on phosphorylated CREB in rat cerebellum: An immunohistochemical study Maria Antonietta Casua,*, Carla Pisua, Angela Sannaa, Simone Tambaroa, Gabriele Pinna Spadaa, Raymond Mongeaua, Luca Pania,b a

Neuroscienze PharmaNess S.C.A.R.L., Via Palabanda, 9 09125 Cagliari, Italy b Institute of Neurogenetics and Neuropharmacology, C.N.R., Cagliari, Italy Accepted 13 April 2005 Available online 23 May 2005

Dedicated to the memory of Anna Porcella, a wonderful friend and scientist who died in September 2003. Her work on CREB and cannabinoids was the basis of our study.

Abstract Several converging lines of evidence indicate that drugs of abuse may exert their long-term effects on the central nervous system by modulating signaling pathways controlling gene expression. Cannabinoids produce, beside locomotor effects, cognitive impairment through central CB1 cannabinoid receptors. Data clearly indicate that the cerebellum, an area enriched with CB1 receptors, has a role not only in motor function but also in cognition. This immunohistochemical study examines the effect of D9-tetrahydrocannabinol (D9-THC), the principal psychoactive component of marijuana, on the levels of phosphorylated CREB (p-CREB) in the rat cerebellum. Acute treatments with D9-THC at doses of 5 or 10 mg/kg induced a significant increase of p-CREB in the granule cell layer of the cerebellum, an effect blocked by the CB1 receptor antagonist SR 141716A. Following chronic D9-THC administration (10 mg/kg/day for 4 weeks), the density of p-CREB was markedly attenuated compared to controls, and this attenuation persisted 3 weeks after withdrawal from D9-THC. These data provide evidence for the involvement of cerebellar granule cells in the adaptive changes occurring during acute and chronic D9-THC exposure. This might be a mechanism by which D9-THC interferes with motor and cognitive functions. D 2005 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Drugs of abuse: opioids and others Keywords: Cannabinoids; CREB; Cerebellum; Immunohistochemistry

1. Introduction Cannabinoid receptors CB1 are expressed at a very high density in the cerebellum, an area of the brain implicated in motor coordination, so it is not surprising that in humans cannabinoids such as D9-tetrahydrocannabinol (D9-THC), the principal psychoactive component of marijuana, have complex effects on psychomotor function. In mice, direct

* Corresponding author. Fax: +39 70 924 2206. E-mail address: [email protected] (M.A. Casu). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.04.053

injection of synthetic cannabinoids into the cerebellum produces motor impairments in the rotorod test, and these deficits are no longer seen in animals that have cerebellar injections of an antisense oligonucleotide directed to a sequence coding for CB1 receptors [11]. Although it has long been known in clinical neurology as much as in experimental neuroscience that the cerebellum is essential for the co-ordination of movement, a growing body of evidence has also implicated the cerebellum in diverse higher cognitive functions. For example, patients with cerebellar diseases have impaired spatial cognition, executive dysfunctions with difficulties in planning, abstraction and working

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memory [29]. These observations raise the possibility that the cerebellar mechanisms implicated in learning and memory might also be relevant in the mechanism of action of cannabinoids. The intake of marijuana induces, beside locomotor effects such as hypolocomotion, ataxia and catalepsy, clear cognitive impairments [32]. Prolonged marijuana usage disrupts short-term memory, working memory, attentional skills and memory retrieval [4]. Administration of cannabis extracts also causes long-lasting memory deficits in rodents, and similar deficits are produced by either D9-THC, endogenous cannabinoids or different synthetic CB1 cannabinoid receptor agonists [6,18,19,21,31]. Several of the molecular and cellular adaptations involved in addiction are believed to be also implicated in learning and memory. Of particular interest is the activation of the cAMP pathway and CREB-mediated transcription which have been related to learning and long-term potentiation of synaptic transmission [35]. Numerous CNS processes, including neurotransmitter synthesis, gene expression and cellular proliferation, are controlled by neurotransmitters acting through second messenger systems that phosphorylate the transcription factor CREB (cyclic AMP response elementbinding protein). CREB is an ubiquitously expressed protein regulated by several intracellular pathways that binds to specific DNA sequences (named CREs or cAMP-response elements) in the regulatory regions of target genes [24]. The transcriptional activity of these dimers is stimulated upon phosphorylation of CREB at Ser133 by several protein kinases, including protein kinase A, Ca 2+/calmodulin-kinase II and IVand several kinases in the mitogen-activated proteinkinase cascade (MAPK). Thus, CREB represents a site of convergence where diverse signaling pathways and their associated stimuli produce plasticity by altering gene expression [30]. Cannabinoids may exert their effects on brain and behavior by modulating signaling pathways controlling gene expression. Accordingly, studies have demonstrated that D9-THC induces the expression of the immediate-early gene c-fos [20]. Furthermore, acute administration of D9THC in rats induces a progressive and transient activation of extracellular signal-regulated kinase (ERK) in the dorsal striatum, nucleus accumbens and hippocampus [10,33]. It has recently been reported that acute D9-THC increases components of the ERK pathway (ERK, p-CREB and c-fos) in the rat cerebellum, while repeated treatment with D9-THC prevents this acute effect of D9-THC [28]. Given that learning, memory and drug addiction share some intracellular signaling cascade events dependent on the activation of CREB [25] and the relationships existing between cognitive deficits, the cerebellum and the adverse effects of cannabinoids, it was important to better understand the effect of D9-THC on CREB in the rat cerebellum. It was still unknown where in the cerebellum an acute D9THC-induced response might be displayed. Furthermore, it was crucial to know whether chronic D9-THC would alter levels of activated CREB a day or several weeks after the

last administration of D9-THC. To this aim, we performed an immunohistochemical study on the cerebellum of acutely or chronically treated rats using an antibody raised against phosphorylated CREB (p-CREB), the activated form that binds to DNA.

2. Materials and methods 2.1. Animals Adult male Sprague –Dawley albino rats (Charles River, Italy) were housed in groups of 5 in standard plastic cages with water and food ad libitum. The animal facility was under a 12:12 h light– dark cycle, constant temperature of 22 T 2 -C and relative humidity of 60%. All experimental protocols were performed in strict accordance with the E.C. regulation for care and use of experimental animals (CEE No. 86/609). The appropriate concentration of D9-THC (purchased as a 10 mg/ml in ethanol solution, from Sigma, St. Louis, Missouri, USA) was prepared by evaporating the alcohol with nitrogen and emulsifying the residue with Cremophor, ethanol and saline (1:1:18). For the acute treatment, rats received vehicle (n = 10) or D9-THC at the doses of 2.5 (n = 10), 5 (n = 10) and 10 mg/kg i.p. (n = 10) and were sacrificed 90 min after for immunohistochemistry. A separate group of rats (n = 5) received an acute injection of SR 141716A (1 mg/kg) 15 min before the treatment with D9-THC or its vehicle. For chronic treatments, rats (n = 12) were injected intraperitoneally with D9-THC once a day for 4 weeks at a dose of 10 mg/kg and were sacrificed 12 h (n = 6) or 3 weeks after (n = 6). Control animals (n = 6) were given vehicle for the same time. D9-THC-treated animals presented typical behavioral cannabinoid effects such as a reduction in spontaneous locomotor activity. 2.2. Immunohistochemistry Rats were perfused transcardially 90 min after D9-THC treatment with 4% paraformaldehyde in 0.2 M phosphate buffer (PB). The brains were subsequently cryoprotected overnight with a solution of 30% sucrose in 0.1 M PB at 4 -C. Alternate sagittal sections of 40 Am were cut at sledge (Microm HM 400 R). Adjacent sections were processed for Nissl staining (cresyl violet from Sigma) or p-CREB immunohistochemistry. For the immunohistochemistry, controls were performed by subtracting the primary antibody in the procedure. The staining was performed as previously reported [8]: after rinsing in phosphatebuffered saline with 0.2% Triton X-100 (PBS + T), sections were incubated with 0.3% of H2O2 in PBS and, after extensive washing, with a blocking solution containing 1% BSA and 20% normal goat serum in PBS + T to reduce background.

M.A. Casu et al. / Brain Research 1048 (2005) 41 – 47

Sections were incubated overnight at 4 -C with a rabbit anti-P-CREB antibody (1:500; Cell Signaling Technology). After rinsing, sections were incubated with an anti-rabbit biotinylated IgG (1:200; Vector, Burlingame, CA, USA) for 1 h followed by an avidin – biotin complex (1:400; Vectastain ABC kit, Vector) for an additional hour. After washing, sections were exposed to 3,3V-diaminobenzidine (0.06% in PBS) containing 1% cobalt chloride and 1% nickel ammonium sulfate for 15 min. Immunostaining was developed by adding 5 Al H2O2 (0.1% in PBS) to each 500 Al of 3,3V-diaminobenzidine. After washing in PBS + T, all sections were mounted on gelatin-coated glass slides, airdried, dehydrated in ascending concentrations of ethanol, cleared with xylene and coverslipped with Entellan. The mounted sections were examined under a BX-60 Olympus light-microscope (Olympus Optical, GmbH, Hamburg, Germany) at 20 and 40. 2.3. Quantitative analysis The analysis was performed in the granular layer of the cerebellar cortex between bregma coordinates 0 and 3.4 mm away from the midline [26] corresponding to the paramedian lobule as the most uniform staining was found in this area. The analysis was carried out using an image analysis system (KS 300; Karl Zeiss Vision GmbH, Hallbergmoos, Germany) by choosing for each section three randomly chosen fields, in at least ten alternate sections for each animal, and by measuring the percentage of the area occupied by p-CREB staining with respect to a 2000 Am2 standardized area (% IM area percentage). Gray values, measured in immunostaining negative areas of the sections, were subtracted as background from the resulting binary picture. All data were expressed as mean T SEM and analyzed using a one-way analysis of variance (ANOVA). When a significant interaction ( P < 0.05) was found, the Newman– Keuls post-hoc test was used. 2.4. Protein extraction and Western blot analysis Rats were killed, and the cerebella were rapidly removed, dissected and placed on an ice-cold plate. Total cell protein extract and Western blot were performed as previously described with some modifications [9]. In brief, tissue samples were homogenized at 4 -C by an homogenizer system (GlasCol, Terre Hante, IN, USA) in 100 Al of 20 mM HEPES buffer (pH 7.9) containing in mM: NaCl, 125, MgCl2, 5; glycerol, 12%; ethylenediaminetetracetic acid (EDTA), 0.2; Nonidet P-40, 0.1%; dithithreitol (DTT), 5; phenilmethylsulphonil fluoride (PMSF), 0.5; leupeptin, 0.5 Ag/ml; and pepstatin A 0.7 Ag/ml. The extracts were then centrifuged at 15,000g (at 4 -C) for 20 min, and the resulting supernatant was collected as total cell extracts. An aliquot was analyzed for protein concentration by using a Protein assay kit II (Bio-Rad Laboratories, Hercules, CA, USA), and the rest was frozen at 80 -C until assayed.

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Aliquots of cerebellar extracts containing 50 Ag of total protein were separated by sodium dodecyl sulfate-polyacrylamide gels (SDS PAGE) and transferred to nitrocellulose membranes (Bio-Rad). Blots were blocked with 5% nonfat dry milk in TBST (0.1% Tween 20 in Tris borate saline) and probed with a specific antibody against Phospho MAPK 1/2 (Cell signaling Technology, Beverly, MA) with a 1:1000 dilution in 1% BSA TBST. After washing in TBST, blots were probed with a horseradish-peroxidase-conjugated antibody with a 1:2000 dilution in TBST plus 5% milk and, after washing in TBST, chemiluminescence was detected by West Pico chemiluminescent substrate (Pierce, Rockford, IL). Immunoreactive bands were visualized with a Fuji Las 1000 image analyzer (Raytest Isotopenmessgera¨te GmbH, Straubenhartd, Germany), and the optical density of immunoreactive bands was measured using a specific software (AIDA 2.11, Raytest Isotopenmessgera¨te GmbH, Straubenhartd, Germany). Student’s t test was performed as a statistical analysis using GraphPad Prism program (San Diego, CA, USA).

3. Results The purpose of the present study was to examine whether acute treatment (2.5, 5 and 10 mg/kg) or chronic treatment (10 mg/kg/day  28 days) with D9-THC affects p-CREB immunostaining (p-CREB-IM) in the rat cerebellum and, if it does, to determine where in the cerebellar cortex this effect of D9-THC occurs. As shown in Figs. 1A – B, in the cerebellar cortex of untreated rats, intense p-CREB immunoreactive cells were found in the granular layer of the paramedian lobule, which contains numerous densely packed small granule cell neurons, while very few stained cells were found in the molecular layer. No positive staining was present in the omission control (without the primary antibody; Fig. 1C). This staining typology was present in all lobules of the cerebellar cortex. Adjacent sections were stained with Nissl to examine the complete neuronal population, and based on this analysis, it was clear that only background levels of pCREB-IM were found in areas such as the Purkinje cell layer (Figs. 1D –E). There were no apparent changes in the distribution patterns in any of the acutely treated groups. However, a marked increase of p-CREB-IM was observed in the granular layer of rats acutely treated with D9-THC (Fig. 2). The administration of D9-THC at doses of 5 and 10 mg/kg i.p. produced an almost 50% increase of p-CREB-IM (one-way ANOVA F 3,36 = 21.77, P < 0.01). The lower dose of D9-THC (2.5 mg/kg) did not modify the density of pCREB-IM (Fig. 2E). The increase of p-CREB-IM after the acute treatment with D9-THC was completely blocked by pretreatment with the CB1 receptor antagonist SR 141716A (Fig. 2F), suggesting that it is mediated by CB1 receptor activation. Note that SR 141716A at a dose of 1 mg/kg did not have any effect by itself on p-CREB-IM (Fig. 2F). Since

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Fig. 1. Micrographs of p-CREB-IM in the paramedian lobule of the rat cerebellar cortex. Note (arrows), at low (A) and high (B) magnification, that p-CREB is present in the granule cell layer (CBXg) but not in either the dense white matter layer (DWL) or the molecular layer (CBXm). Negative control experiment done in absence of CREB antibody incubation showed no staining in the granular layer (C). Notice, at high magnification, the p-CREB positive granule cells and the absence of p-CREB-IM in Purkinje cell layer in view of the sections stained with Nissl (D – E). Scale bars A – C = 100 Am; B = 50 Am; D – E = 20 Am.

a small quantity of ethanol was added in the vehicle and considering that ethanol can increase p-CREB levels [34], we compared rats treated with vehicle with rats treated with saline, but we did not find any difference between these two groups (vehicle = 14.2 T 5; saline = 12.68 T 2% immunostained area). Since the phosphorylation of CREB may be mediated by the MAPK pathway through the phosphorylation of ERK, we focused our attention on the D9-THC effect on the pERK induction using a Western blot approach. An increase of p-ERK expression in the cerebellum of rats treated with D9-THC compared to controls (147.3 T 15.72 vs. control 100 T 10; n = 8; P < 0.05) has been found. This result suggests that CREB phosphorylation may be induced by the activation of the MAPK pathway, one of the proposed signaling cascades regulated by CB1 receptor activation. For the chronically treated rats, an additional control was made since the withdrawal group was not handled to receive an injection the day before. No difference in p-CREB levels was found between rats that were injected with vehicle the day before and rats that were not handled for 3 weeks (nonhandled: 12.6 T 1.8; handled: 14.8 T 3.2% immunostained area). Furthermore, there was no apparent change in the distribution pattern of p-CREB-IM in the cerebellar cortex between rats chronically treated with vehicle or D9-THC (Figs. 3A –B). The histogram of Fig. 3D shows that a chronic treatment with D9-THC at a dose of 10 mg/kg/day for 4 weeks produced a small but significant decrease (about 35%) of p-CREB-IM in the cerebellar granular layer with respect to vehicle-treated rats (one-way ANOVA F 2,15 =

3.98, P < 0.05). Interestingly, the decrease of p-CREB-IM, observed 4 weeks after treatment with D9-THC, persisted 3 weeks after withdrawal of D9-THC (Figs. 3C – D).

4. Discussion Our interpretation of the present results is in line with the generally accepted view that transcription factors such as CREB are implicated in synaptic plasticity underlying learning, memory and addiction [24]. Regulation of gene expression is one mechanism that explains relatively stable changes within neurons. Exposure to drug of abuse would eventually lead to changes in nuclear function and to altered rates of transcription of particular target genes by causing repeated perturbation of intracellular signal transduction pathways. Altered expression of these genes would lead to altered activity of the neurons in which those changes occur and, ultimately, to changes in the function of neural circuits where neurons operate. Like other G-protein-coupled receptors, CB1 receptors are linked to multiple intracellular signal transduction pathways that phosphorylate CREB, such as for example the cAMP, the MAPK and the ERK pathways [23]. Recently, it has been demonstrated that acute D9-THC administration induces a progressive and transient activation of ERK [10,28,33], a protein kinase known to be critical in synaptic plasticity and memory [1,13], in several brain areas including the cerebellum. The latest study [28] using both Western blot and ELISA techniques has shown that acute D9-THC injection (15 mg/kg) increases the

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Fig. 2. Density of p-CREB-IM in the paramedian lobule of the rat cerebellar cortex following acute D9-THC administration: (A) micrograph showing pCREB-IM following vehicle administration; (B – D) micrographs showing p-CREB-IM following D9-THC at the dose of 2.5, 5 and 10 mg/kg, respectively. (E) Histogram illustrating the percentage of the area covered by p-CREB-IM in the granular layer of treated rats. (F) Antagonistic effect of SR 141716A pretreatment (1 mg/kg, i.p.) on p-CREB-IM induced by acute D9-THC administration (5 mg/kg) in the rat cerebellar granular layer. Significant increases in p-CREB-IM were found at doses of 5 and 10 mg/kg compared to vehicle-treated animals (**P < 0.01). Scale bar in micrograph D = 200 Am.

expression of various protein factors of the ERK pathway, including p-ERK itself, p-CREB and FOS. The results of the present immunohistochemical study are consistent with the finding that acute D9-THC increases p-CREB in the

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cerebellum. Furthermore, using Western blot analysis, we confirmed in the present study that acute D9-THC also increases the expression of ERK. These results suggest that the ERK pathway contributes to the initiation of a gene expression program in response to cannabinoids by activating CREB, as was reported for other addiction drugs such as cocaine and opiates [14]. The present study indicated that acute administration of D9-THC induces a significant increase in the levels of the pCREB-IM in the granular layer of the cerebellar cortex. It is interesting that the augmentation of p-CREB following D9THC exposure did not occur uniformly throughout the cerebellum but was most apparent in the glutamatergic granule cell layer. It is important to underlie that the granule cells axons ascending into the molecular layer form the parallel fibers that make connections with Purkinje cell dendrites [2,3,16]. Long-term depression of synaptic transmission at the level of parallel fiber to Purkinje cell synapse (a unique and characteristic type of synaptic plasticity in the cerebellum) is though to play an essential role in motor learning [17]. Conceivably, cannabinoids effects on CREB in granule cells might alter plasticity at the level of Purkinje cells synapses. The increase of p-CREB following acute treatment with D9-THC may be linked with a cannabinoidinduced event occurring from membrane components of signal transduction at granule cells terminals in the molecular layer where cannabinoid CB1 receptors appear to be mainly located [12]. We observed a decreased density of p-CREB-IM in the cerebellum following a long-term treatment with D9-THC, and this effect persisted in animals that were withdrawn from D9-THC for 3 weeks after a chronic treatment. These results indicate that the levels of p-CREB are depressed following repeated D9-THC administration, and this condition persists even in the absence of D9-THC in the bloodstream (note that because the density of un-phosphorylated CREB was not measured, it is not known at this point whether this decrease in p-CREB is due to a decrease in the phosphorylation of CREB or a decrease in the expression of the CREB protein). In view of our findings, it is possible that this apparent desensitization of the D9-THC-induced pCREB results from the lower basal levels of p-CREB occurring after repeated D9-THC administration. It was not the objective of the present study to assess a desensitization of CB1-mediated p-CREB response by testing the effect of an acute challenge with D9-THC in animals chronically treated with this drug. Nevertheless, it is important to bear in mind that other studies support the notion of a desensitization/down-regulation of CB-1 receptors following long-term treatment. Chronic treatment with D9-THC (10 mg/kg, 21 days) significantly reduced the net WIN 55,212-2-stimulated [35S]GTPgS and cannabinoid receptor binding in most brain regions including the cerebellum, suggesting not only a reduction in the number of cannabinoid receptors, but also a marked desensitization of cannabinoid-activated signal transduction mechanisms after chronic D9-THC [5].

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Fig. 3. Density of p-CREB-IM in the paramedian lobule of the rat cerebellar cortex following a chronic D9-THC treatment (4 weeks) and 3 weeks after withdrawal: (A) micrograph showing p-CREB-IM following vehicle administration; (B) following a chronic D9-THC treatment at a dose of 10 mg/kg/day; (C) following a chronic D9-THC treatment at a dose of 10 mg/kg/day followed by a 3-week withdrawal period. (D) Histogram illustrating the percentage of the area covered by p-CREB-IM in the granular layer of rats chronically treated with D9-THC at a dose of 10 mg/kg and after 3 weeks of withdrawal. Significant decreases in p-CREB-IM were found after the chronic treatment and also after the 3-week withdrawal, compared to vehicle-treated animals (*P < 0.05). Scale bar in micrograph A = 200 Am.

Our finding that chronic D9-THC lowers p-CREB levels is informative in view of the fact that chronic marijuana consumption in humans causes memory deficits [4]. Learning was shown to be associated with an increase of hippocampal p-CREB in rats [7], while CREB-deficient mice display deficits of contextual memory [15]. Persistent low levels of p-CREB following repeated D9-THC exposure, such as that found in the cerebellum, could thus explain the memory deficits caused by this drug. In further studies, it would be interesting to find out whether there is a correlation between low levels of p-CREB in animals chronically treated with D9-THC and a decrease behavioral performance in cerebellum-dependent memory tasks. In fact, it has been demonstrated, using the behavioral model of eyeblink conditioning, that both the hippocampus and the cerebellum constitute a brain circuitry that mediates recently acquired memory. Furthermore, cerebellar damage induces a specific behavior in radial maze tasks, characterized by an inflexible use of the procedure and by a severe impairment in working memory processes [22]. Our finding that the decrease of p-CREB-IM persisted over 3 weeks after the last drug injection points to cellular mechanisms that could mediate long-term morphological changes in the cerebellum following repeated cannabis exposure. During neural development, p-CREB is a critical factor that determines cerebellar cytoarchitecture by regulating the differentiation of granule cell neurons [27]. Prolonged alterations in p-CREB levels might thus have profound effects on cerebellar circuitry. These questions require further investigation since there are no data about the effect of repeated cannabis exposure on cerebellar morphology.

In conclusion, acute D9-THC administration increases pCREB levels mostly in the granule cell layer of the cerebellum, while long-term exposure to that drug persistently depresses p-CREB levels in that same area. It is suggested that this latter phenomenon, by interfering with aspects of cerebellar plasticity and morphology, accounts for some of the adverse effects of cannabinoids exposure on the brain, such as memory impairments.

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